Standard Conditional Knockout
A standard conditional knockout is a versatile tool for studying your gene of interest – there are as many possible patterns of knockout as there are Cre lines.
The schematic below illustrates the targeting strategy of a conditional knockout mouse model in which exon 2 was flanked with loxP sites, and the Neo cassette was introduced into the intron downstream of exon 2. Upon FLP and Cre recombination, exon 2 is eliminated, and a loxP/FRT footprint remains.
The deletion of exon 2 results in a frame shift and early stop codons. The mRNA produced from this disrupted gene is targeted by the nonsense-mediated decay pathway, generally resulting in no protein being produced. As illustrated below it is also possible for a truncated protein product to be made before early stop codons are encountered.
Wildtype protein sequence as encoded by exon 1-4:
Protein sequence after deletion of exon 2 (as encoded by exons 1, 3 and 4):
(Frameshift mutation and predicted stop codons denoted by asterisks and yellow highlighting)
A specific target sequence within a gene of interest is flanked by loxP sites to facilitate Cre recombinase-mediated excision, for tissue specific or temporally controlled gene inactivation.
The following general considerations apply for conditional knockout mouse models and are evaluated by our experts for each conditional knockout model generated at ingenious.
Preservation of wildtype expression before Cre recombination
- Avoid insertion of exogenous sequences into regulatory regions.
- Avoid disruption of promoter elements.
- Avoid disruption of splice sites.
- Insert genetic elements into large introns.
Inactivation of the gene after Cre recombination
- Target the earliest exon that will result in a frameshift and premature stop codons upon deletion.
- Target an important domain.
- Delete as much coding sequence as possible.