Abstract
Purpose :
To create mouse models to study eye stiffness in Glaucoma. We have previously shown that the inhibitor of calcification/ stiffness Matrix gla (Mgp) gene is highly expressed in the trabecular meshwork (TM) and peripapillary sclera (ppSC) in a Mgp knock-in mouse model. Because Mgp knock-out (KO) mice are lethal, here we set out to generate a Mgp-loxP mouse line which would allow the creation of TM & ppSC conditional KOs. We further investigated CRISPR/CAS9 strategies for the generation of floxed mice
Methods :
Our CRISPR/CAS9 strategy involved: 1) search for optimal insertion sites for the loxP sequences in the Mgp gene, presence of nGG sequences and absence of conserved or repeated regions 2) generation of 2 guides RNA (gRNA) by in vitro transcription containing sequences homologous to the insertion site and binding to Cas9 3) generation of recombinant Cas9 protein 4) generation of a donor exogenous DNA containing loxP sequences (37 nt) flanked by restriction sites and homologous arms 5) co-injection of the 2 gRNAs, Cas9 protein and donor vector into one cell embryo and implantation on the foster mother 6) screening of pups by Southern Blot (SB)
Results :
loxP sites were inserted to flank Mgp exons 3 and 4, at 330 bp 5’ of exon 3 and 893 bp 3’ of exon 4. Predicted excision leads to a non-functional Mgp protein. To assure better integration and insertion in cis, the loxP sites were flanked by 5’ 1,100 bp and 3’ 705 bp homology arms (donor vector). The efficiency of gRNA-mediated cleavage was tested in vitro by incubating the gRNAs, Cas9 protein and a WT target site PCR fragment. A total of 69 eggs were injected with the gRNAs/Cas9/donor DNA mix and 21 were implanted. Six founder pups were born. F1 pups screened by SB resulted in the expected 2.9 kb (5’ probe), 3.7 kb (3’probe) and 2.1 kb (internal probe). F2 pups were identified as WT, loxP/+ or loxP/loxP by PCR with 2 sets of primers yielding 505 bp (5’loxP), 324 bp (3’loxP) and 264 bp (WT) fragments. Mgp-floxed mice are fertile and have normal life span (6 months at submission)
Conclusions :
The CRISPR/CAS9 strategy presented here has been successful in generating the first reported Mgp-floxed mouse line. The availability of this mouse is an essential requirement to generate Mgp conditional KOs in TM and ppSC, and it would be invaluable for the creation of stiffness mouse models in glaucoma
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.