Knockin Mouse Model Generation
Applied StemCell offers custom knockin mouse model generation service using two complementary technologies, CRISPR/Cas9 and TARGATT™ site-specific integration technologies that vastly expand the portfolio of knockin modifications we can offer gene-specific knockin (up to 6 kb) or safe harbor locus knockin (up to 10kb with CRISPR and up to 22 kb with TARGATT™). We offer several genetic modifications: point mutation, humanized gene knockin/ replacement, reporter mouse models, safe harbor locus models with CRISPR (Rosa26, H11, AAVS1) or TARGATT™ (Rosa26, H11 or a custom locus).
Case Study# 1: CRISPR Knock-in Model - Generation of site-specific 2 kb large fragment knock-in mouse using CRISPR/Cas9
Goal: To insert a 2 kb large fragment DNA (gene of interest) at “a specified locus” in the mouse genome using CRISPR/Cas9.
The project was designed using a well optimized protocol to generate the transgenic mice: (1) Cas9 mRNA and gRNA were produced by in vitro transcription; (2) donor vector was constructed by in-fusion method: the plasmid contained 1 kb long 5’ and 1 kb long 3’ homologous arms flanking the gene of interest (2kb); (3) the mixture of Cas9 mRNA, gRNA and donor vector was microinjected into fertilized eggs of C57BL/6j mouse background.
Using a panel of genotyping primer pairs, three out of 31 pups born after microinjection (#15, 19, and 26) were identified as founders (F0), with the gene of interest inserted at the desired locus.
Figure 1: Agarose gel electrophoresis of PCR results in F0 mice (#15, 19, and 26) with site-specific gene knock-in. The left part of the gel shows the 5’ junction fragment (2,191 bp), and the right part of the gel shows the 3’ junction fragment (2,557 bp). [wt: wildtype control; M: 1 kb DNA ladder].
Case Study# 2: CRISPR Knock-in Model - Site-specific knock-in of a 27 bp tag in mice using CRISPR/Cas9
Figure: Five (#2, 5, 6 13 and 17) out of nineteen pups, 26%, were genotyped by restriction enzyme digest and sequencing to confirm the correct knock-in on one of the alleles (heterozygotes). A sequence with desired knock-in generated a different cutting pattern compared to its wild type counterpart.
