Construction and identification of primary open angle glaucoma disease MYOC Pro356Leu mutant plasmid in mouse

PURPOSE: To construct and identify the plasmid of mutant MYOC Pro356Leu in mouse.

METHODS: 1.Transfering Red recombinantinase- expressing plasmid pKD46 was transferred to RP23-180F15 BAC clone by electroporation.2.Amplifying tThe pStart-K vector was amplified by PCR and electroporating the PCR product was electroporated into the bacteria containing the mMYOC. 3.PCR amplification of the mMyoc AfeI fragment and Subcloningand then subcloned it into pBluescript vector.4.Designing two oligonucleotides was designed and introducingthe Pro356Leu (C to T at codon 356) was introduced into the pBS_mMyoc AfeI clone. 5. the Introducing pBS_mMyoc AfeI mut clone was introduced to pStart-K_mMyoc by digestion and ligation reaction. Identifying correct the final clones were identified by restriction enzyme digestion and sequenceing.

RESULTS: pStart-K_mMyoc mut clone,the mouse MYOC Pro356Leu mutant plasmid was constructed and identified well by restriction enzyme digest and sequenceing.

CONCLUSIONS: It is a good method to construct a long DNA fragment clone by red recombinase and pStart-K vector. The constructed Mouse MYOC Pro356Leu plasmidwill be very helpful for the studies on the function and biologic effects of MYOC gene in primary open angle glaucoma.