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Scientists Can Analyze Gene Function by Deleting Gene Sequences

An illustration shows a mouse standing on a surface. The mouse's fur is white with black spots. Its ears, nose, four paws, and tail are tan. The mouse is sitting on its haunches with its front paws in the air.
One way to understand the function of a gene is to observe a biological system that lacks that gene. But what is the best system to use? When studying human genes, researchers typically employ biological systems that approximate these genes and their functions as closely as possible. In particular, researchers often turn to mice because of all the various model organisms most commonly used in the lab (e.g. fruit flies and yeast), mice have the genome that most closely resembles that of humans. Consequently, manipulation of genes within the mouse genome has proven an effective method for learning about human gene functioning. Indeed, experimentally removing or altering certain genes within a mouse allows for the examination of a biological system with specific gene alterations.

What can mice reveal about human gene function?

On the outside, humans and mice look nothing alike. However, human and mouse chromosomes share many of the same genes. In fact, 99% of the 20,000 to 25,000 genes in humans have a similar mouse counterpart. This high degree of genetic similarity between humans and mice offers researchers a unique approach for understanding human gene functioning.

Ethical considerations prevent researchers from genetically engineering humans that lack a given gene for the sole purpose of learning how that gene functions. However, because so many mouse genes are similar to human genes, geneticists can generate knockout mice in which the mouse counterpart of a human gene of interest is deleted or disrupted. The term "knockout mouse" may at first conjure images of a mouse boxing champion or beauty queen. However, geneticists use the term to refer to a mouse that has been genetically engineered such that at least one of its genes is functionally inactivated (i.e. the inactivated gene is "knocked out"). If a mouse gene has a high degree of similarity to a human gene, researchers can predict that the two genes carry out related functions. Therefore, by generating a knockout mouse without a gene of interest, these scientists may be able to determine the functions carried out by the related human gene.

In some cases, knockout mice exhibit phenotypes that mimic symptoms associated with human disease, including cancer, diabetes, obesity, cardiovascular disease, and neurodegenerative disease. In these cases, a knockout mouse is referred to as a mouse model of that form of human disease.

Making a knockout mouse: Step by step

How are genes deleted or disrupted in a knockout mouse? In the past, making a knockout mouse was a major undertaking. Today, however, a combination of new molecular biology techniques and increased knowledge of the mouse genome has reduced the time and effort required to make a knockout mouse. Nevertheless, there still are a number of steps involved in engineering one of these creatures.

Step 1: Generating a targeting vector

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.

An illustration shows a region of DNA containing a target gene. The DNA is depicted as a horizontal grey cylinder, separated into three equal segments. The center segment is the target gene; the segments on either side of the gene are homologous DNA segments 1 (left) and 2 (right). A second illustration shows the insertion of the Neo and TK genes. Neo is depicted as a yellow rectangle and confers neomycin resistance. TK is depicted as a green square and confers ganciclovir sensitivity. The Neo and TK genes are inserted into the target gene sequence to make a targeting vector sequence. The resulting targeting vector is now composed of, from left to right: homologous DNA segment 1, the neomycin resistance gene, homologous DNA segment 2, and the TK gene.
Figure 1: The targeting vector is designed to contain both neomycin-resistant and ganciclovir-sensitive (TK) sequences.

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.

Step 2: Inserting the target sequence and selecting cells with the insertion

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?

A schematic diagram shows the fate of a targeting vector after its insertion into ES cells via electroporation.
Figure 2: The targeting vector is inserted into the ES cell genome, and disables the target gene.

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.

Step 3: Identifying ES cells with the correct gene knocked out

An illustration shows a cell culture dish containing 14 spherical cells scattered randomly in the dish. Seven of the cells are red; the remaining seven cells are yellow. The cells are being grown in a light pink culture medium.
Figure 3: In a cell culture dish, only a portion of ES cells will contain the targeting vector. These \"knockout ES cells\" will survive exposure to neomycin and ganciclovir (shown in red), while the other cells will die (pink). The surviving cells will then be used for the next step.
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.

Step 4: Injecting heterozygous knockout ES cells into a developing embryo and transferring the embryo into a mouse

An illustration shows the injection of three knockout ES cells into a mouse embryo. The embryo is depicted as a ball composed of at least 17 yellow and red circles. Three red circles, representing ES cells, are being injected into the ball with a triangle-shaped syringe.
Figure 4: Knockout ES cells are then injected into a fresh mouse embryo containing normal cells.
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).

An illustration shows a mouse standing in an erect position on a flat surface, its snout in the air. The mouse's fur is white with black spots. Its ears, nose, four paws, and tail are tan.
Figure 5: A chimeric mouse contains both normal cells and genetically manipulated \"knockout\" cells. Coat color can reflect this with a spotted pattern.
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.

Step 5: Mating chimeric mice to yield homozygous knockout mice

An illustration shows a genetic cross between two mice, and the resulting progeny mouse. A solid white mouse mates with a white mouse with black spots. The resulting mouse is a solid black color.
Figure 6: Mating a chimeric (spotted) mouse with a normal (white) mouse can yield a knockout mouse, among other normal mice. All mice born from this mating can be screened to verify the gene has been knocked out.
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.
A schematic shows the steps involved in designing a knock-out mouse. Each step is accompanied by illustrations and a text description, and separated by process arrows. Step 1 describes the design of a target vector using the Neo and TK genes. Step 2 describes the insertion of the target vector into ES cells using electroporation. Recombination of the target gene with the target vector produces cells that are resistant to neomycin and ganciclovir. In step 3, only the cells that have successfully incorporated the targeting vector into the target gene survive in the presence of neomycin and ganciclovir. In the illustration, a cell culture dish contains 14 spherical cells. Seven of the cells are red; the remaining seven cells are yellow. The red cells represent those cells that have successfully incorporated the targeting vector. In step 4, cells containing the targeting vector are then selected and injected into a normal developing mouse embryo. In step 5, the resulting chimeric spotted mouse contains a mix of its own cells and the heterozygous knockout cells. This mouse is bred with a normal white mouse. Among their offspring are mice that are capable of passing the knocked out gene to their own offspring.
Figure 7: The steps involved in making a knockout mouse.

Step 6: Phenotypic characterization of homozygous knockout mice

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).

Large-scale mouse knockout projects

In September 2006, the U.S. National Institutes of Health (NIH) initiated a five-year, $52 million project called the Knockout Mouse Project (KOMP). Together with the Texas A&M Institute of Genomic Medicine (TIGM), the North American Conditional Mouse Mutagenesis Project (NorCOMM) in Canada, and the European Conditional Mouse Mutagenesis Program (EUCOMM), KOMP set the goal of knocking out every one of the 20,000 mouse protein-encoding genes within five years.

Similar to the mouse knockout consortia mentioned above, a number of labs have also collaborated to establish standardized methods for the phenotypic characterization of knockout mice. The European Union Mouse Research for Public Health and Industrial Applications (EUMORPHIA) group developed the first set of standard phenotyping protocols, which was validated among several different labs. The European Mouse Phenotypic Resource for Standardized Screens (EMPReSS) then established the primary phenotypic screen used by the European knockout mouse labs, one that comprises a subset of the standard protocols of EUMORPHIA.

Future directions

Future studies of knockout mice will continue to yield new and unexpected discoveries regarding the function of mouse genes and their human counterparts. An increased understanding of human gene function will also lead to the design of more effective treatments for human disease.

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