15.8:
In-vitro Mutagenesis
Scientists can inactivate or knock out a gene in animals, commonly mice, to learn more about the function of the gene, usually by replacing it with a targeting vector, an engineered piece of DNA.
The targeting vector has sequences that are homologous, identical to the sequences before and after the gene. It usually also has a positive selection marker such as the gene for neomycin resistance NeoR in the middle and a negative selection marker like the gene for thymidine kinase TK at one end.
When introduced into embryonic stem cells it replaces the gene through homologous recombination which occurs naturally between stretches of DNA with similar sequences. Cells where the gene has been correctly replaced will contain the positive marker but not the negative one allowing them to be identified in culture. These cells are then inserted into a mouse embryo and implanted into the uterus of a female. The resulting mouse has a combination of normal cells and cells with the gene knocked out on one chromosome.
These mice are then bred to generate so-called knockout mice that are homozygous for the knockout in all cells.
15.8:
In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
The Process
Genes can be randomly knocked out, or specific genes can be targeted. To knock out a particular gene, an engineered piece of DNA called a targeting vector is used to replace the normal gene, thereby inactivating it.
Targeting vectors have sequences on each end that are identical—or homologous— to the sequences flanking each side of the gene of interest. These homologous sequences allow the targeting vector to replace the gene through homologous recombination—a process that occurs naturally between DNA with similar sequences during meiosis.
The targeting vector is introduced into mouse embryonic stem cells in culture, using methods such as electroporation—use of electric pulses to temporarily create pores in the cell membrane. Typically, to identify cells where the vector has properly replaced the gene, it is designed to include a positive selection marker—such as the gene for neomycin resistance (NeoR)—between the homologous regions; and a negative selection marker—such as the gene for viral thymidine kinase (TK)—after one of the homologous regions.
The cells are exposed to neomycin, and only those that have incorporated the vector into their DNA will survive because they have the NeoR gene. Also, cells, where the vector has replaced the targeted gene through homologous recombination, will not have the TK gene, allowing them to survive in the presence of the drug ganciclovir. Therefore, exposure to ganciclovir is used to eliminate cells that have the vector randomly inserted into their genome, because these cells will have the TK gene.
The cells with the gene properly knocked out are then inserted into a mouse embryo, which is implanted into the uterus of a female, where it develops until birth. The resulting mouse is a chimera—meaning it is composed of a mixture of cells—some with normal DNA from the embryo, and some with the gene knocked out on one chromosome from the engineered cells. These mice are bred, and offspring containing the gene in their germline are further crossbred to create a line of mice where every cell is homozygous for the knockout. These knockout mice can then be used to study gene function.
Suggested Reading
Hall, Bradford, Advait Limaye, and Ashok B Kulkarni. “Overview: Generation of Gene Knockout Mice.” Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino … [et Al.] CHAPTER (September 2009): Unit-19.1217. [Source]