A transgenic mouse contains additional, artificially-introduced genetic material in every cell. This often confers a gain of function, for example the mouse may produce a new protein, but a loss of function may occur if the integrated DNA interrupts another gene. A transgenic mouse a very useful system for studying mammalian gene function and regulation because analysis is carried out on the whole organism.
Transgenic mice are also used to model human diseases that involve the overexpression or misexpression of a particular protein.
Transgenic mice contain extra genetic material integrated into the genome in every cell.
The extra genetic material is often described as foreign DNA, but it can come from any source, including another mouse.
To get the same foreign DNA sequence into every cell of the mouse, it is necessary to introduce the DNA into cells of the very early mouse embryo that will contribute to the germ line (the cells that produce eggs or sperm).
How does it work?
There are two major methods.
In 'pronuclear microinjection', the foreign DNA is introduced directly into the mouse egg just after fertilization. Using a fine needle, the DNA is injected into the large male pronucleus, which is derived from the sperm. The DNA tends to integrate as many tandemly-arranged copies at a random position in the genome, often after one or two cell divisions have occurred. Therefore, the resulting mouse is only partially transgenic. If the transgenic cells contribute to the germ line, then some transgenic eggs or sperm will be produced and the next generation of mice will be fully transgenic.
The second method is the introduction of DNA into embryonic stem cells (ES cells). These are derived from the very early mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo.
DNA introduced into ES cells may integrate randomly, as in the case of pronuclear microinjection. However, if the introduced DNA is similar in sequence to part of the mouse genome, it may undergo 'homologous recombination' and integrate as a single copy at a specific site (see knockout mice).
ES cells will colonize a host embryo and often contribute to the germ line, resulting in the production of some sperm carrying the extra DNA. When these transgenic sperm fertilize a normal egg, a transgenic mouse is produced with the same foreign DNA in every cell.
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There are two major methods for producing transgenic mice. The first method, 'pronuclear microinjection', begins by isolating eggs just after they have been fertilized.
How is it used?
In most cases the addition of foreign DNA to the genome results in a gain of function, such as the production of a new protein or the _expression of an existing protein at a higher level or in a different range of cells. This is a generally useful approach for studying gene function or regulation, but can also be used to model human diseases caused by dominantly acting mutant proteins (such as Alzheimer's disease).
The addition of foreign DNA can also cause loss of function if it interrupts or disturbs the _expression of an existing gene. This is one strategy used to generate knockout mice.
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 a structural gene, the DNA usually includes other sequences to enable it
to be incorporated into the DNA of the host and
to be expressed correctly by the cells of the host.
Transgenic sheep and goats have been produced that express foreign proteins in their milk.
Transgenic chickens are now able to synthesize human proteins in the "white" of the eggs.
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.
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
can be infected by polio virus and even
develop paralysis and other pathological changes characteristic of the disease in humans.
Two methods of producing transgenic mice are widely used:
transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA;
injecting the desired gene into the pronucleus of a fertilized mouse egg.
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
the structural gene you desire (e.g., the insulin gene)
vector DNA to enable the molecules to be inserted into host DNA molecules
promoter and enhancer sequences to enable the gene to be expressed by host cells
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
Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
Transfer the embryos into her uterus.
Hope that they implant successfully and develop into healthy pups (no more than one-third will).
6. Test her offspring
Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10-20% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain
Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene.
Mating these will found the transgenic strain.
The Pronucleus Method (Method "2")
1. Prepare your DNA as in Method 1
2. Transform fertilized eggs
Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
Inject the male pronucleus with your DNA.
When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.
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 structural gene for human growth hormone
a strong mouse gene promoter
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
restores function in a mutant animal or
knocks out the function of a particular locus.
In either case, targeted gene insertion requires
the desired gene
neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells;
tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog gancyclovir. DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills cells that contain the tk gene.
Treat culture of ES cells with preparation of vector DNA.
Most cells fail to take up the vector; these cells will be killed if exposed to G418.
In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but killed by gancyclovir.
In still fewer cells: homologous recombination occurs. Stretches of DNA sequence in the vector find the homologous sequences in the host genome and the region between these homologous sequences replaces the equivalent region in the host DNA.
Culture the mixture of cells in medium containing both G418 and ganciclovir.
The cells (the majority) that failed to take up the vector are killed by G418.
The cells in which the vector was inserted randomly are killed by gancyclovir (because they contain the tk gene).
This leaves a population of cells transformed by homologous recombination (enriched several thousand fold).
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:
Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. The mouse genome appears to have sufficient redundancy to compensate for a single missing pair of alleles.
Most genes are pleiotropic. They are expressed in different tissues in different ways and at different times in development.
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:
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. [Link to technique]
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 mRNAs.
Successfully-transformed cells were then
fused with enucleated sheep eggs [Link to description of the method] and
implanted in the uterus of a ewe (female sheep).
Several embryos survived until their birth, and two young lambs have now 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.
grow faster than sheep 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.
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 (e.g. lysozyme) in egg white.
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.
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 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.