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According to the IMPC, more than half of knockout mice die during embryo. In order to study the function of these genes during development and adult, conditional knockout mice are particularly necessary. Conditional knockout is genetic damage that occurs in a particular time and in a particular cell and is invaluable for studies of the onset, progression, and progression of many diseases. The most commonly-used system for conditional knockout is Cre/loxP, which takes about a year or more. The CRISPR/Cas9 tool has become very popular because of its ease of use and convenience. And the CRISPR/Cas9 tool has proven its worth in the development and research of conditional knockout mice. With the help of CRISPR Cas9 technology, the solution to conditional knockout mouse models is now more practical than ever and produces better results.
CRISPR/Cas9 PlatformCB is a leading biotechnology company specializing in CRISPR/Cas9 gene-editing services, with an experienced research team, national laboratory. Based on our platform, CRISPR/Cas9 PlatformCB provides you with a professional conditional knockout mouse model service that enables you to have precise control over where or when your gene of interest is knocked out. Using the CRISPR and Cre/LoxP systems, we can generate a floxed conditional knockout mouse model for various time and space knockout conditions in mice. We guarantee that at least 2 founders or 3 F1 animals are born for gene knockout.
Workflow of Conditional Knockout Mouse Services Using CRISPR and Cre/LoxP
A single stranded donor DNA (ssDNA) is used for delivering the floxed targeted exons of the gene you interest, to replace the wild type form. The ssDNA contains two LoxP sequences respectively located at 5' and 3' homologous arms. Cas9 binds to gRNA, and causes targeted double-strand breaks (DSBs) in genomic DNA, ssDNA acts as a DNA repair template that will lead to the two LoxP sequences insert into the targeted genes.
And finally, we obtain a founder mouse with loxP sequences inserted into the appropriate position of the gene of interest.
Cre-recombinase will target at the LoxP sites and catalyzes the deletion of floxed targeted exons. As Cre expression is driven by a selected promoter: a cell-specific or ubiquitous promoter, the Cre-mediated floxed targeted exons deletion is in a cell-specific manner or not, causing a frameshift mutation in the downstream sequence. CKO mice are mated with mice that specifically expressed Cre, and their progeny are genotyped to obtain F1 mice with loxP sequence and Cre enzyme, tissue-specific knockout mice. In addition, CKO mice can be mated with Cre-ERt2 deleter mouse to generate F1 mice with loxP sequences and Cre-ERT2, conditionally knockout mice. Cre-ERT2 is temporarily triggered by an external inducer-agents (usually by small molecules such as tamoxifen or tetracycline), and knock out the gene of interest at a particular time point.
Typically, we develop CRISPR-mediated knockout mice in C57BL/6 and FVB, but we can use other mouse strains according to your requirements.
CRISPR/Cas9 PlatformCB, as a leading biological commercial company, is dedicated to providing professional CRISPR/Cas9 gene-editing services. We have state-level laboratories that meet standard animal feeding conditions and experienced scientists. Tell us the gene you interest, we will provide you with customizing CRISPR/Cas9 Conditional Knockout Animal Model services from gRNA design to F1 mice. In addition, we will provide you with a final report, which details each step, including target vector design, construction and validation, transfection conditions, genotyping strategies and results. Don’t hesitate to contact us, we will meet your specific requirements and protect your scientific investment.