Construction Of A Mouse Model Expressing ?C31 Integrase

. As one of the most important research fields in modern biotechnology, transgenic animals not only has great value in basic researches on gene function and developmental studies but also has huge profit on application researches in agriculture, medicine and pharmaceutical industry. Traditionally, the main method for generating transgenic animals is pro-nuclear microinjection. However, this approach has some disadvantages such as low integration rates and poor expression for position effect of foreign genes.Streptomyces?C31 integrase is kind of recombinases, which can mediate integration of a donor plasmid containing attB sequence and foreign gene into endogenesis specific sequence(pseudo attP)in many species. Moreover, the utilization of?C31 integrase can provide long-term and high levels foreign gene expression. To solve the obstacles caused by pro-nuclear injection, this study constructs a mouse model expressing?C31 integrase. Two kinds of?C31 integrase expression mouse have been obtained. One is the?C31 integrase driven by EF1?promoter, which comprises EF1?-Int and EF1?-Int-NLS and have?C31 integrase ubiquitously expressed. Another is the?C31 integrase driven by Zp3 promoter, which comprises pZP3-INT and pZP3-INT-NLS and have?C31 integrase expressed restrict in oocyte. EF1?-Int mice were further investigated.?C31 integrase mRNA was detected in the zygotes of EF1?-Int mice by RT-PCR analysis. Afterwards, we microinjected pBCPB+ into the zygotes of EF1?-Int mice and PCR results indicated the recombination of pBCPB+, generating a new attL site, which demonstrated that?C31 integrase in zygotes could mediate the recombination between attB and attP sites.Additionally, a fluorescent binary switch was developed to evaluate the function of?C31 integrase in living cells expediently. .In this switch system, the red fluorescent protein (RFP) expresses in the absence of?C31 integrase but the green fluorescent protein (GFP) is produced when?C31 integrase are expressed and catalyzes a recombination event. . To test the validity of this system, NIH3T3 cells were transiently transfected with different ratios of the plasmid encoding?C31 integrase and the?C31 integrase report plasmid, and the proportion of cells with red or green fluorescence were quantitatively measured by FACS. The numbers of cells converted from red to green increased with the increase of amount of?C31 integrase encoding plasmid cotransfected. Approximately 90% of the color alternation was reached when the ratio of?C31 integrase encoding plasmid to reporter plasmid was 10:1. This result indicated that the?C31 integrase could mediate the deletion of reporter plasmid in the context of genomic. Thus, this system may also be useful for producing transgenic mouse as“reporter”mice for?C31 integrase. We can construct a mouse model by targeting the?C31 integrase reporter into a ubiquitously expressed genomic site via homologous recombination in embryonic stem cells. Therefore, the?C31 integrase reporter mouse, which could be a fluorescent binary switch when?C31 integrase is present, will be a useful tool for lineage-tracing studies.In conclusion, this study constructed a mouse model with?C31 integrase expression that can be used to generate transgenic mice with site-specific integration. This could offer an alternative way of producing transgenic mice with site-specific integration of foreign genes to avoid the disadvantage of the random integration in traditional pro-nuclear injection method.. Moreover, we constructed a dual fluorescent system to evaluate the function of?C31 integrase in living cells. Meanwhile, this system provides elementary experimental data for producing?C31 integrase reporter mice.