Floxed allele can be created for tissue- or temporal- specific deletion of your gene of interest. This can be very useful when studying embryonic lethal genes or for understanding how your gene of interest functions in different tissues. CRISPR/Cas9 is used to insert loxP sites flanking the region to be deleted.
This line can then be crossed with a Cre-line expressing the Cre recombinase under a specific promoter or injected with a plasmid containing Cre recombinase. The Cre recombinase promotes recombination of the two loxP sites and the region between the sites is removed from the genome.
One of the more difficult aspects of biology is how to perform loss-of-function studies of essential genes?
- Used to make deletion of genes in site and time specific manner
- Flank gene of interest with LoxP sites
- Inject a plasmid or cross into strain that expresses Cre recombinase where/when/how you want loss-of-function to occur
We are able to build custom Cre recombinase-expressing lines to compliment your new floxed allele.
Insertion of two loxP sites to produce a knockout (KO) of an embryonic lethal gene
A client wanted a knockout (KO) of an embryonic lethal gene. We could not make this line using our standard methods. Instead, we inserted two loxP sites. One in the first intron of the gene and the second in the 3’utr. After we confirmed this line by PCR and sequencing, we injected this line with a ubiquitously expressing Cre Recombinase plasmid.
We found while the uninjected animals could reproduce, the Cre injected animals did not (Figure A). We tested recombination by PCR and found that only the Cre injected progeny showed recombination of the loxP sites (Figure B).
|Fig. A||Fig. B|
This line could be used to study KO of this gene in adulthood or in specific tissues, something that was not previously possible due to the embryonic lethality caused by the KO of this gene.
Service Details (price reflects academic pricing)
|Service Package||Price||Est. Delivery Time|
|Full Build||$4,485||6 - 8 Weeks|
|Candidate Lines||$2,964||3 - 4 Weeks|
|Custom Injection Mix||$1,395||1 - 2 Weeks|