Scientists Can Analyze Gene Function by Deleting Gene Sequences

The first step in making a knockout mouse is identifying the region of the gene that will be deleted. Because the entire mouse genome sequence is already known, it is relatively simple to look up the chromosomal location and nucleic acid sequence of the gene of interest. Once the segment of the gene that will be deleted has been mapped out, the nucleic acid sequences of the DNA segments that appear on the chromosome before and after that gene must also be identified.

Once these tasks are completed, a targeting vector specifically tailored to the gene of interest is made (Figure 1). A targeting vector is a long stretch of DNA made up of smaller pieces of DNA that have been joined together. Next, in order to make the targeting vector detectable, the scientists insert a marker gene into the middle of the vector; this marker is in some way able to "report" when it is present in a cell.

Currently, the neomycin-resistance gene, called NeoR, is a popular marker gene of choice for generating knockout mice. The antibiotic neomycin is toxic to mouse cells because they do not normally contain the NeoR gene. However, when the NeoR gene is added to mouse cells, these cells can survive in the presence of neomycin. Within the targeting vector, the NeoR gene is located between two other pieces of DNA: the "right arm" and the "left arm" of the targeting vector. The right arm of the targeting vector contains DNA with a nucleic acid sequence that matches the stretch of DNA immediately before the gene segment that will be deleted. The left arm of the targeting vector contains DNA with a nucleic acid sequence that matches the stretch of DNA immediately after the gene segment that will be deleted. The right and left arms of the targeting vector facilitate homologous recombination between the targeting vector and the target gene, thereby enabling the NeoR gene to replace the target gene segment.

The targeting vector also contains one additional piece of DNA, called a negative selection marker gene. This gene is located at the right end of the targeting vector, after the right arm. The thymidine kinase (TK) gene from the herpes simplex virus is the most commonly used negative selection marker gene. Normally, mouse cells can grow in the presence of the antiviral drug ganciclovir. The TK gene is considered a "cell suicide gene," however, as cells containing the TK gene convert ganciclovir into a lethal toxin.

Why is it necessary to include a cell suicide gene as part of the targeting vector? The reason is purely a matter of identification - specifically, the TK gene helps researchers locate cells that have correctly replaced the targeted gene segment with the NeoR gene. Often, mouse cells randomly insert the targeting vector in the wrong chromosomal location. If random insertion occurs, both the NeoR gene and the TK gene are inserted into the genome. As a result, the cells are resistant to neomycin, but they die in the presence of ganciclovir. In comparison, when the targeted gene segment is correctly replaced, the TK gene is not inserted into the chromosome along with the NeoR gene, so the resultant cells are resistant to both neomycin and ganciclovir. Therefore, the presence of the TK gene in the targeting vector allows researchers to efficiently screen for mouse cells that have correctly replaced the targeted gene segment by growing these cells in the presence of both neomycin and ganciclovir.

After the targeting vector is made, it is used to knock out one copy of the target gene in mouse embryonic stem (ES) cells (Figure 2). But why must mouse ES cells be used? Why can't the targeting vector be introduced into any type of mouse cell?

It is certainly possible to use the targeting vector to knock out one of the two copies of the target gene in a standard somatic cell. However, unless that cell is an ES cell, the knockout mutation cannot be incorporated into a growing embryo. Therefore, it would not be possible to study the effects of the knockout mutation in a developing mouse.

What makes ES cells so special? Primarily, it is their ability to become any one of the different adult cell types. When injected into a mouse embryo, the ES cells themselves are capable of maturing into some of the tissues of the developing mouse.

But how, exactly, are targeting vectors delivered into ES cells? Most often, a technique called electroporation is used. When ES cells are electroporated, a brief pulse of an electrical field is applied to the outside of the cells, creating a momentary increase in plasma membrane permeability and allowing the uptake of foreign DNA into the ES cells.

After the ES cells have been electroporated, they are grown in the presence of neomycin to select for those particular cells that have taken up the targeting vector. Next, the neomycin-resistant cells are grown in the presence of ganciclovir to select for those that have inserted the targeting vector at the correct location within the mouse genome.

Additional experiments using standard molecular biology techniques help researchers determine whether the target gene has been fully knocked out in the ES cells that are resistant to both neomycin and ganciclovir. After these experiments are complete, only the ES cells that have had one copy of the target gene knocked out remain (Figure 3). These ES cells are heterozygous for the knockout mutation. Although these cells will grow and divide in culture, they cannot form an embryo that will develop into a mouse on their own.

How, then, do the heterozygous knockout ES cells grown in culture develop into a mouse? The first step involves injection of these cells into a developing mouse embryo (Figure 4). This step allows the heterozygous knockout ES cells to become part of the developing embryo. Then, because they are ES cells, the heterozygous knockout cells are incorporated throughout the embryo and are capable of becoming any type of tissue within the developing mouse. Therefore, like a patchwork quilt, the developing embryo contains a mixture of its own original cells and the heterozygous knockout cells. Because of this cellular mixing, the resultant mouse is called a chimeric mouse (Figure 5).

How can researchers tell whether the heterozygous knockout cells have been incorporated into the mouse embryo? And how can the knockout cells be distinguished from the original mouse cells? The primary means for determining the success of knockout cells has to do with the coat color of the mice involved. The original ES cells that are electroporated with the targeting vector come from two parents with black coats, which is the dominant coat color in mice. In contrast, the original cells of the developing embryo come from two parents with white coats, which is the recessive coat color. When the heterozygous knockout cells are successfully incorporated into the developing embryo, the resulting mouse will have patches of heterozygous knockout cells and patches of original cells, so its coat will have both black and white patches of fur.

Still, only some of the cells that make up the chimeric mouse carry the knockout mutation, and those cells are heterozygous for the mutation. In order to learn about the function of the target gene, a mouse that is homozygous for the knockout mutation must be studied.

How can researchers use chimeric mice to produce homozygous knockout mice? The answer lies in the reproductive tissues of chimeric mice, some of which are made up of heterozygous knockout cells. As a result, some of the gametes from chimeric mice carry knockout mutations. To produce a mouse that is homozygous for the target gene knockout, chimeric mice capable of passing the knockout mutation on to their offspring must be identified.

These chimeric mice can be identified by crossing them with normal white mice. If black offspring are produced from such a cross, a chimeric mouse is capable of passing the knockout mutation on to its offspring. When this is the case, 50% of the black offspring are heterozygous for the knockout mutation in all of their cells (Figure 6).

Standard molecular biology techniques can be used to determine which of the black offspring are heterozygous for the knockout mutation. Then, to produce a homozygous knockout mouse, a heterozygous knockout male is mated to a heterozygous knockout female (Figure 7). Twenty-five percent of the resulting offspring will be homozygous knockout mice, which can, again, be readily identified using standard molecular biology techniques.

After the homozygous knockout mice have been created, researches must then characterize the phenotypes associated with the loss of the target gene. In theory, every measurable phenotype must be examined in order to determine every possible function of the knocked-out gene. The measurement of a given phenotype in a knockout mouse would then be compared to measurements of the same phenotype in a wild-type mouse (a mouse that has not been genetically engineered for specific traits) in order to identify the functions that are altered in the knockout mouse.

Phenotypes such as size, weight, metabolism, behavior, bone development, neurological function, reproduction, and aging can be easily measured. If the knocked-out gene is required for development, however, it may not be possible to produce homozygous knockout mice. In this case, researchers may study heterozygous knockout mice, or they may instead turn to other types of knockout mice, such as conditional knockout mice (in which the target gene is inactivated in response to a specific stimulus) or tissue-specific knockout mice (in which the target gene is inactivated in only one or several tissues).