Hipp11 CAG-rox-stop-rox-td-sfGFP dre reporter mouse

Hipp11 (aka Igs2) knock-in of a dre / rox reporter with td-sfGFP (tandem dimer superfolder green fluorescent protein).

Generated with a standard workflow in the Capecchi laboratory: RPCI-23 C57BL/6J BAC library clone recombineering to generate the targeting vector; electroporation of the G4 B6 × 129 F1 hybrid mouse embryonic stem cells.

The targeting vector was deposited at Addgene, and they sequenced it in its entirety. The NGS sequence is available at https://www.addgene.org/139513/sequences/. One can verify that the homology arms in the targeting vector map to Hipp11 using NCBI BLAST.

In addition to the targeting vector, empty backbone plasmids with the homology arms that directed high efficiency knock-in into Hipp11 by embryonic stem cell electroporation were also deposited (http://www.addgene.org/browse/article/28203395/). The NGS sequences of these plasmids are available at http://www.addgene.org/126649/sequences/ and http://www.addgene.org/127054/sequences/.

As one can see, in ES cells, the combination of the Hipp11 locus and the CAG promoter resulted in very high expression level.

With the germline mice, I could homozygose the Hipp11 knock-in allele after repeated backcrosses to the C57BL/6J mice, meaning no wildtype allele PCR product was amplified in those mice using a standard 3 primers set up. Together with the Southern blots, this is evidence good as any that the intended locus was initially knocked in. Of course, there is whole genome sequencing, but for now, that is beyond my budget.

Video of C57BL/6J backcrossed homozygous Neo+ Hipp11 reporter mice. The Neo+ Hipp11 reporter mouse line was backcrossed to the C57BL/6J background for total of 14 generations then intercrossed. Shown here is a cage with an F1 homozygous female and her F2 pups from a cross with an F1 homozygous male.

Video of C57BL/6J backcrossed homozygous ΔNeo Hipp11 reporter mice. The ΔNeo Hipp11 reporter mouse line was backcrossed to the C57BL/6J background for total of 16 generations then intercrossed. Shown here is a cage with an F1 homozygous male and his F2 pups from a cross with an F1 homozygous female, prior to cage changing.

Acknowledgment to Dr. Anne Boulet for taking the photographs and movies in my absence (because I left Salt Lake City on 4/22/2023).