Development Of CMV-GFP Transgenic Mice And Observation Of Expression Of The Transgene

Objective: To develop CMV-EGFP transgenic mice and observe the expression oftransgene under the regulation of CMV promoter.Method: After digesting the plasmid pEGFP-N1 with two restriction enzymes——Ase I,AflⅡ,the 1.6kb DNA fragment was purified and recovered and dilutedinto a final concentration 3.2ng/ul.The purified DNA fragment were injectedit into the pronucleus of the one-cell fertilized eggs of B6D2F2.When theembryos injected developed into 2-cell,some were cultured in vitro to observethe expression of the gfp transgene under the control of CMV promoter.Theothers were reimplanted into the oviducts of psudopregnant KM femailmice.Seven days after the pups were born,they were observed under UV lightto determine wether they were green.When the pups were 3 weeks old,the genomicDNA were extracted from the tail tips as the models of PCR and Southern blottingto check if the pup was positive transgenic animal.Result: In total, 3264 fertilizated eggs were injected,1779 of which developedinto 2-cell embryos after in vitro cultured.The survival rate was 54.50%.1397survived embryos were reimplanted into the oviducts of psudopregnant kunbaifemale mice.After reimplantation,16 female mice were pregnant and 85 pups wereborn. PCR analysis indicated 11 out of 85 pups weretransgenic.SouthernBlotting detection showed 1 out of 11 was transgenic.The other 382 embryoswere cultured in vitro.19 embryos emited green fluorescence when they wereobserved under fluorescent microscope,4 of which were homozygotes and 15 werechimeras.Conclusion: Positive transgenic mice can be developed via pronuclearmicroinjection. Under the regulation of CMV promoter,gfp transgene can beexpressed in the early stage of embryo,but can not always be expressedwidespreadly in the whole embryo.