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Vector Design
iTL generates its targeting constructs using the high fidelity Red/ET recombineering method. We isolate genomic BACs from our internal library and use homologous recombineering to produce the vectors. Compared to the traditional PCR based process, BAC recombineering is a stronger and more robust technique less likely to introduce unwanted mutations.
The targeting vector creation process includes analysis of gene structure, screening strategy design, probe design and validation, isolation and confirmation of genomic BAC clone, subcloning of homology arms, insertion of loxP &/or FRT flanked PGK Neo cassette, construct validation and preparation of DNA for electroporation.
iTL Delivers:
Conventional targeting vector: A target region of interest is replaced with a Neomycin selection cassette allowing precise selection during tissue culturing and resulting in inactivation of the target gene(s).
Conditional knockout vector: The target region is flanked by loxP sites which facilitate Cre-recombinase mediated excision of the target region in a tissue-specific or temporally controlled manner.
Knockin point mutation(s): Required mutations are engineered to replace targeted base(s) with alternate amino acid(s). Different methods can be combined with the use of loxP or other recombineering sites that enable the mutations to be active/inactive in specific tissues or at different developmental timelines.
Knockin human/murine cDNAs or reporter genes: A variety of reporter genes are available for replacement of an endogenous gene to facilitate expression studies. Reporters include GFP, LacZ, mCherry, tdTomato, YFP or DsRed. They can also be fused to the N or C terminus of your gene. If co-expression of the reporter is required we offer the use of IRES or 2A peptide strategies.
Targeted transgenic: A cDNA is inserted into a specific locus allowing control by the endogenous promoter and regulatory elements.
