Mouse Transgenesis By Intracytoplasmic Sperm Injection

In our previous study, normal and fertile mice were successful produced from oocytes following intracytoplasmic sperm injection (ICSI) . In the present thesis, the possibility of producing transgenic embryos and offspring with this procedure was evaluated. After freezing-thawed once using HEPES-CZB medium without cryoprotectants, the cauda sperm from KM fertile male were exposed to the circular or linear pEGFP-Nl DNA for 1 min and then co-injected into metaphase II oocytes of B6D2F1 strain. When the zygotes with two pronuclei were cultured in CZB medium to day 3.5, 39.1%(9/23) of them, derived from oocytes co-injected with sperm head and pEGFP-Nl plasmid DNA, were expressed GFP protein. After transfer of the ICSI embryos with two pronuclei from co-injection of sperm head and foreign DNA, seven recipients delivered 30 pups (23.8%, 30/126) . Southern blot results revealed that three of sixteen offspring integrated with GFP and neomycin genes together(18.8%) . Interestingly, all of them were produced from oocytes co-injected sperm head and linear DNA (33.3%, 3/9) , while none of seven ICSI offspring integrated either GFP or neomycin gene in the group of co-injection of sperm head and circular plasmid DNA. Southern blot analysis also revealed that the foreign gene could be transmitted by germ lines. These results indicated that the high efficiency of transgenic mouse could be produced by ICSI. It may be shown that linear DNA is more easily to integrate into host genome than linear DNA when ICSI was used to produce transgenic animals.