Construction Of A Mouse Model Expressing φC31 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.StreptomycesφC31 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 ofφC31 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 expressingφC31 integrase. Two kinds ofφC31 integrase expression mouse have been obtained. One is theφC31 integrase driven by EF1αpromoter, which comprises EF1α-Int and EF1α-Int-NLS and haveφC31 integrase ubiquitously expressed. Another is theφC31 integrase driven by Zp3 promoter, which comprises pZP3-INT and pZP3-INT-NLS and haveφC31 integrase expressed restrict in oocyte. EF1α-Int mice were further investigated.φC31 integrase mRNA was detected in the zygotes of EF1α-Int mice by RT-PCR analysis. Afterwards, we microinjected pBCPB+ into the zygotes of EF1α-Int mice and PCR results indicated the recombination of pBCPB+, generating a new attL site, which demonstrated thatφC31 integrase in zygotes could mediate the recombination between attB and attP sites.Additionally, a fluorescent binary switch was developed to evaluate the function ofφC31 integrase in living cells expediently. .In this switch system, the red fluorescent protein (RFP) expresses in the absence ofφC31 integrase but the green fluorescent protein (GFP) is produced whenφC31 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 encodingφC31 integrase and theφC31 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 ofφC31 integrase encoding plasmid cotransfected. Approximately 90% of the color alternation was reached when the ratio ofφC31 integrase encoding plasmid to reporter plasmid was 10:1. This result indicated that theφC31 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 forφC31 integrase. We can construct a mouse model by targeting theφC31 integrase reporter into a ubiquitously expressed genomic site via homologous recombination in embryonic stem cells. Therefore, theφC31 integrase reporter mouse, which could be a fluorescent binary switch whenφC31 integrase is present, will be a useful tool for lineage-tracing studies.In conclusion, this study constructed a mouse model withφC31 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 ofφC31 integrase in living cells. Meanwhile, this system provides elementary experimental data for producingφC31 integrase reporter mice.