Abstract: 【Objective】 In order to get a solid foundation for the site selection of a gene targeting experiment which aimed to realize the co-expression of the foreign gene and the endogenous gene linked through 2A sequence, the effect on the gene expression of different location of the loxp sequence at the open reading frame of a gene need to be clarified when the Cre/loxp system was selected as a tool for reporter gene deletion. 【Method】The enhanced green fluorescent protein (egfp) gene was selected as experimental gene, and red fluorescent protein (dsred2) gene as internal reference gene. Based on pEGFP-N2, pGEM-5zf-loxp plasmid, three plasmids with different location of loxp sequence at the open reading frame of egfp gene named ploxp-EGFP (The loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region of egfp gene), pEGFP-loxp (The loxp sequence located at the downstream of the termination codon in the 3′un-translation region of egfp gene) and ploxp-EGFP-loxp (There is one loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of egfp gene) were constructed. As the internal reference, Plasmid pDsRed2-N1 was co-transfected with constructed plasmids into PK15 cells. Twenty-four hours later, the fluorescence intensity of every transfection was observed and analyzed using fluorescence microscope and software Image J. In order to verify the result obtained using egfp gene as experimental gene, phytase gene was selected as another experimental gene. Based on pIREsNeo, pGEM-5zf-loxp, pT-phytase, pGL4.13[luc2/SV40] plasmid and luciferase gene as expression internal reference gene four plasmids with different location of loxp sequence at the open reading frame of phytase gene named p-SV40-luciferase-CMV-loxp-phytase (The loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region of phytase gene, p-SV40-luciferase- CMV-loxp-phytase-loxp (There is one loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of phytase gene), p-SV40-luciferase-CMV- phytase-loxp (The loxp sequence located at the downstream of the termination codon in the 3′un-translation region of phytase gene), p-SV40-luciferase-CMV-phytase (There is no loxp sequence located at the upstream of the Kozak sequence in the 5′un-translation region and another downstream of the termination codon in the 3′un-translation region of phytase gene)were constructed. As transfection internal reference, pEGFP-N2 was co-transfection with the differenct plasmid constructed above into PK15 cells, pEGFP-N2 and pDsRed2-N1 were co-transfected into PK15 cells to work as blank control for enzyme activity of phytase and luciferase. Forty-eight hours later, the fluorescence intensity of every transfection was observed and analyzed using fluorescence microscope and software Image J. These data were analyzed using statistics software SPSS19.0. At the same time, the activity of phytase and luciferase of every transfection were measured by Molybdenum Blue Method or using Firefly Luciferase Assay Kit, and these data were analyzed using statistics software SPSS19.0.【Result】In the experiment, the dsred2 gene worked as internal reference gene. Statistics analysis using the average value of red fluorescence of different area of every transfection as basic data shows there is no significant difference between every different transfection. This result shows there is no significant difference in purity of plasmid, handling and cell activity between every transfection. Plasmid electrophoresis result shows there is no significant difference in quality between every plasmid used in transfections, this means the difference in quality between every plasmid used in every transfection is not enough to influence the accuracy for analysis. Analysis on the green fluorescence in the same rule shows there is no significant difference between different transfection of the same plasmid and between pEGFP-N2 and pEGFP-loxp, ploxp-EGFP and ploxp-EGFP-loxp, but there is significant difference between pEGFP-N2, pEGFP-loxp and ploxp-EGFP ploxp-EGFP-loxp. These result shows loxp sequence had no effect on egfp gene expression when it is located downstream of the open reading frame of the gene, but had a negative effect on egfp gene expression when it located upstream of the open reading frame of the gene. Replacing the egfp gene by phytase gene and using egfp gene as transfection internal reference gene, luciferase gene as expression internal reference gene, it shows a same result.【Conclusion】These results indicate that if we try to prepare a transgenic animals in which the foreign gene and the endogenous gene is linked by 2A sequence and co-express under the regulation of the full regulation element of the endogenous gene in one copy manner and the Cre/loxp system is selected as a tool for reporter gene deletion, the downstream of the open reading frame of the endogenous gene is the suitable targeting site.

Key words: loxp sequence, gene expression, 2A sequence, gene targeting