Deleting a relevant mammalian gene constitutively (in the germ line) or conditionally (in certain tissues or upon intervention) is a way to investigate its malfunction or its potential as a drug target in a variety of disease areas – in neurodegeneration, cancer, CVD, and many other fields.
Why Constitutive Knockouts
The simplest approach for elucidating the elementary function of a gene is its inactivation.
Since the advent of easily accessible techniques for conditional knockouts in mice, constitutive KO’s were more rarely generated. Currently, we generate constitutive knockouts predominantly for species that are less easily accessible – rats and rabbits. Here, a constitutive knock out is possible via nuclease based engineering (CRISPRs, TALENs, etc.).
Constitutive Knockout Strategies
There are several way of disrupting gene function. In constitutive approaches and via homologous recombination, we may target and excise exon 1; or a downstream exon, if the precise initiation exon is not certain. Protein structure and coding frames must be considered. Careful designs always include careful supporting analysis of hidden genomic elements that should be left intact.
The gene of interest or the relevant exon (GOI) is deleted constitutively.
Why Conditional Knockouts
Global inactivation of a gene can sometimes lead to embryonic lethality; or sometimes, mimicking a defaulting gene action is desired by inducing global inactivation in adult age. In other cases, the disruption of a gene only in certain tissues is the goal of a genetic investigation.
Here’s when we target the gene in a manner that leaves it intact and fully functional, but allows for conditional inactivation upon combining with publicly available mouse strains that are pre-validated for this purpose. Conditional targeting also allows to irreversibly (“constitutively”) disrupt the gene in the germ line, upon appropriate breeding.
At PolyGene we do conditional knockouts in mice and rats via the CRISPR/Cas9 technology. Please in inquire for more details.
Conditional Knockout Strategies
The Cre/loxP system is the basis to conditionally cut out a DNA fragment from the mammalian genome. It is a highly reliable and efficient tool in use since the early 1990’s, and users can therefore build on a large library of Cre transgenic mouse lines that have been generated and entered into the public domain.
We flank a region of interest with loxP sites; we do so by targeting that region with a recombination vector containing the loxP sites. Typically, a few relevant exons and up to 2 kbp are flanked. Due to specialized base vectors, construction is almost as easy, and as rapid, as building a constitutive targeting vector.
Flanking the GOI by recombination sites (e.g. loxP, here in red) allows its deletion in a tissue-specific or inducible manner.