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There are many reasons why the issue of conventional vs. conditional knockout mouse models is solved through a clear advantage of conditional methods. Conventional or whole-body knockouts were originally considered the most advanced genetic engineering technology for mouse models some 30 years ago. The latest techniques in use today, however, allow for far greater accuracy and efficiency in studying human disease by allowing conditional deletion or activation of target genes in a manner that better mimics the human condition. Conditional knockout in particular is ideal for ensuring that many of the most common limitations of conventional knockout techniques can be bypassed entirely.
When it comes to comparing conventional vs. conditional knockout methods, it helps to know the basics. While conventional knockouts were developed first and involve animal models with artificially impaired or eliminated genes in all tissues of the body, conditional knockouts are more advanced, involving gene knockouts that only target specific tissues or organs. For example, in order to study a disease that affects the liver, scientists might use a conditional knockout that eliminates a specific gene only in that specific organ. Their results can then become much more accurate in terms of the drugs and genetic therapies developed to help people living with liver disease.
In terms of understanding the drawbacks of conventional vs. conditional knockout models, it’s important to remember that conventional knockout is a knockout that can affect all cell types. Some cells are required for embryonic development and if important cells are affected by gene knockout during embryo development, a mouse model may never come to fruition due to embryonic lethality. Also, the gene of interest will be knocked out at all times and in all tissues where the gene is active with a conventional knockout. This limits studies on what occurs in the mouse during different stages of activation and deactivation of the gene of interest because in the case of a conventional KO, the gene is always inactivated.
The Cre-loxP system is used to study gene function in selected cell types and/or at specified time points. Two mouse lines are required for this system to work. In the first mouse line, loxP sites are inserted into a target gene of interest, in introns and typically flanking a critical region of the gene. Prior to administration of Cre recombinase, this mouse model functions like wild type. Once mated to a second mouse line containing the Cre transgene, Cre recombinase can act on the loxP sites, excising the DNA between the loxP sites to create a gene knockout of the target gene. Tissue- and/or time-specificity is determined by which promoter is driving the Cre transgene. For instance, if Cre is being driven by a lung-specific promoter, then only cells that express the target gene in the lungs will have a knockout of the target gene in the lungs.
When considering conventional vs. conditional knockouts, it’s important to realize that from the start, conditional knockouts had more capabilities than conventional knockouts. With a conditional KO, gene inactivation can occur in a certain tissue type or at a specific time point made possible by mating a loxP mouse with a specific Cre line available in repositories. Traditionally conventional and conditional mouse models were made by manipulating the gene of interest first in embryonic stem cells that would have to be selected and screened for proper targeting, then injected into blastocysts in order to produce live mice. Today, advanced technologies such as CRISPR/Cas9 can be used to inject zygotes directly, eliminating the time-consuming embryonic stem cell work of the traditional approach. Researchers are potentially able to achieve knockout sooner, and sometimes homozygous knockouts can be found right from the start.
Almost a decade after the initial development of the CRISPR/Cas9 technique, this and other advanced technologies for obtaining conditional knockouts have become more popular and widely used than conventional techniques. Newer technologies allow for simplicity and efficiency, diminishing the time required to obtain modified target genes and enhancing the bioinformatics tools of the past to identify appropriate RNA sequences. When it comes to the advantages or disadvantages between conventional vs. conditional knockout models, it’s safe to say that the added flexibility of conditional knockout along with new technological knockout methods favor the easier and more result-driven conditional knockout methods, to the older, traditional techniques of making a conventional knockout.
With a traditional or conventional knockout, a target gene is permanently inactivated in all tissues at all time points, while a conditional knockout allows for the target gene to be turned off at specific times or in specific tissues.
Conditional knockout mice are obtained by crossing a loxP or “floxed” line with a Cre transgenic line. The target gene becomes inactivated in vivo within the expression domain of Cre.
Floxing is the introduction of loxP sequences on either side of a targeted gene. The presence of Cre recombinase leads to deletion of the intervening sequences.
Gene targeting is a method used for creating knockout mice using embryonic stem cells, or ESCs. The homologous recombination process in ESCs is robust and allows for specific gene targeting of large genomic regions.
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