Generation Of CMV-Cre-EGFP Transgenic Mice

Objective: To develop CMV-Cre-EGFP transgenic mice and observe the expression of transgene under the regulation of CMV promoter.Method: After digesting the plasmid pEGFP-N1 with two restriction enzymes——Nsi I,AflⅡ,the purified 3.3kb DNA fragment was recovered and diluted into a final concentration 2.9ng/ul.The purified DNA fragment were injected into the pronucleus of the one-cell fertilized eggs of B6D2F2.When the injected embryos developed to 2-cell stage, some were cultured in vitro to observe the expression of the Cre-EGFP transgene under the control of CMV promoter. The others were implanted into the oviducts of pseudopregnant ICR female mice. Seven days after the pups were born, they were observed under blue-light to determine whether they emitted green fluorescence. When the pups were 3 weeks old, the genomic DNA were extracted from the tail tips as templates of PCR to check if the pup was positive transgenic animal. The positive transgenic mice were crossed with ROSA reporter mice, which carried LacZ gene floxed by LoxP at ROSA loci. The recombination competence of Cre recombinase in their progenies was tested by LacZ staining. Homozygous mice was breeded by genetics methods, and CMV-Cre-EGFP strain was established.Result: In total, 2391 fertilizated eggs were injected, 1500 of which developed into 2-cell embryos after in vitro cultured. The survival rate was 62.74%. 1390 survived embryos were implanted into the oviducts of 73 pseudopregnant ICR female mice. After implantation, 15 female mice were pregnant and 36 pups were born alive. PCR analysis indicated 2 out of 36 pups were transgenic positive, of which one male mouse emitted green fluorescence at whole body by excitation of blue-light. After crossed with ROSA reporter mice, their filial mice represented Cre recombinase activity by LacZ staining. Three homozygous mice were breeded by genetics methods, and CMV-Cre-EGFP strain was established.Conclusion: Positive CMV-Cre-EGFP transgenic mice can be obtained via pronuclear microinjection. Under the regulation of CMV promoter, Cre-EGFP transgene can be expressed in the transgenic mice. And the consequent Cre recombinase efficiently mediated recombinations between two LoxP sites.