Structural Biochemistry/Nucleic Acid/DNA/Knockout Mouse

General Information
A knockout mouse is a mouse used by researchers for laboratory experiments aimed at understanding the consequences of inactivation or "knocking out" of a specific gene. In general, the over- and/or under-expression of genes in an organism for experimentation is known as transgenic technology. This process is completed by disrupting or replacing the existing gene with an artificial piece of DNA that is a mutated version of the targeted gene. Due to the disruption, there will be a loss of gene activity, and it will cause changes in the mouse's phenotype. When the mouse's phenotype is affected, the changes in appearance, behavior, and other physical characteristics should be evident in the offspring.

Purpose and Applications
As many genes are similar in mice and humans, the extraction or "knocking out" of a particular gene in a mouse can provide evidence to further understand the extent of the function of genes in humans. This usually is manifested by a change in the animal's physical characteristics, behavioral characteristics, or biochemical pathways that regulate the mouse's functions. This laboratory technique has been used in various types of research:


 * Cancer Research
 * Cystic Fibrosis
 * Lung, Heart, Blood, and Parkinson Diseases
 * Aging
 * Anxiety
 * Arthritis
 * Diabetes
 * Obesity
 * Neural Pathway Functions
 * Substance Abuse

A specific gene studied from the knockout mouse can also be useful in studying how different recreational drugs affect the animal, which can be used to test therapies for drug abuse in humans. For example, a p53 knockout mouse focuses on a mechanism - p53 - that codes for a protein that inhibits the growth of tumors and stops cell division. By taking out this gene, the mouse is at risk of developing various types of cancer (blood, lung, brain, bone, etc.). This is a useful study because humans with the abnormality in this gene have | Li-Fraumeni Syndrome, a rare autosomal dominant hereditary disorder that puts people at a much higher risk of developing cancer.

Limitations and Weaknesses
Although knockout mouse technology is an excellent research tool, there are frequent complications that occur when a particular gene is knocked out. For example, the mouse might depend on the gene of interest for other important bodily functions; if it was disrupted, the mouse might die or stop functioning correctly in unexpected ways. In addition, the gene that is knocked out in the mouse may not even produce an observable change in any of the mouse's characteristics. This makes it extremely difficult to correlate the study with that of humans. Gene knockouts in mice embryos may sometimes inhibit the mice from growing into adult mice. This makes the studies limited to the pre-natal stage of the mouse, further distancing the relationship between the gene-knockout in the mouse, and that of humans.

Methods of Preparing Knockout Mice


Knockout mice are created from embryonic stem cell (ES cells) by harvesting them approximately 4 days after fertilization. The reason for using the ES cells so early on is because the swapping of gene sequences can be properly passed on to the rest of the cells during division and develop along with the all the other adult cells. This process is completed in one of two methods:

Gene Targeting
In gene targeting, a particular gene is manipulated within the nucleus of the ES cells of the mouse through homologous recombination. To start the homologous recombination, the DNA sequence of the gene that needs to be replaced would need to be known. Next is to make a new DNA sequence that is needed to be inserted into a chromosome. That chromosome is going to take the place of a wild-type allele. The artificial inactive DNA sequence is introduced (this piece is nearly identical in sequence to the knocked out gene). This artificial sequence flanks the DNA sequence in both directions on the chromosome. The cell recognizes the identical stretches of DNA, and "trades" the existing gene with the artificial DNA. Since the artificial DNA is inactive, the function of the existing gene has now been "knocked out" by gene targeting. The new cells will keep growing and dividing with the new gene inside of it.

Example: An embryo from a mice in the blastocyst stage of a species with gray fur is isolated. Then the embryonic stem cells are removed from the blastocyst and put on a tissue culture to be grown. Transfer the homologous recombinant gene and grow them in gancyclvir and neomycin. The cells with the new genes for white fur are then transferred back to the blastocyst. Many of the transformed blastocysts will be implanted into pseudo-pregnant mouse with white fur. The mouse will give birth to some white mice and some with patches of gray, showing the activity of the old gene. The mice with the patches - which means they have both the gray fur and white fur genes - will mate with a white mouse. If the gametes of the gray white mouse were from the recombinant stem cells then it will give birth to all gray mice. All of the cells in the mice are heterozygous for the fur gene. The gray mice will then mate together with the heterozygous mice. Identify which mice has the homozygous recombinant and mate them until both of the alleles are knocked out. The end result is the knock out mouse which is when both of the alleles have been knocked out.

Gene Trapping
Gene trapping is done by using a sequence of artificial DNA which holds a "reporter gene" that is made to insert into any gene at random. The artificial DNA prevents RNA splicing in the cell, thus preventing the existing gene from synthesizing its assigned protein and eliminating its function. Now the activity of the artificial "reporter gene" can be observed and studied, to determine the existing gene's normal function in the mouse.

Which method is better?
For both of these methods, a DNA vector is used to carry the artificial DNA into the embryonic stem cells of the mice. Once the DNA is injected, the cells are cultured in-vitro, and then injected into mouse embryos. These embryos are given planted into female mice, which then give birth to mice with the knocked out genes.

Both ways have their own advantages. For example, in gene targeting, the target gene is known in the DNA sequence. This method allows researchers to knock out the sequence(s) that they find are interesting. On the other hand, although the specific gene which is knocked out is unknown in gene trapping, it would create different kinds of mice because there is no efficiency or precision in how the "reporter gene" binds; finding the function of specific gene can become cumbersome because of the randomness. The researchers need to spend a lot of time testing the ES cell to identify which gene has been knocked out. Moreover, a certain gene that is not easily chosen may be knocked out in random manner.

Resources

 * 1) Wikipedia: Knockout Mouse
 * 2) National Genome Human Research Institute: Knockout Mice