| Conditional gene knockout mice models, developed by Cre/loxP recombinationsystem, are great tools in researching gene functions in vivo. However, mice and ratsare not suitable models for the study of human diseases in certain aspects as they arerodents. Pigs are similar to humans in organ size, structure, and physiology. Thus,developing conditional pig models will be an exciting biomedical feat. In recent tenyears, gene modified pig models, constructed by pig somatic cell nuclear transfer(SCNT), have become a focus in human diseases. In this study, we try to constructtransgenic (Tg) miniature pigs with tissue-specific expression of Cre recombinaseusing pig SCNT. It will be an important method in constructing tissue-specific geneknock miniature pig models.In this study, we selected the porcine AQP2promoter to drive the expression ofCre recombinase in Tg miniature pigs. AQP2(Aquaporin2) is a water channelassociated with renal water excretion and is thought to be preferentially expressed inthe kidney collecting duct. In the current study, we analyzed the expression patterns ofpig AQP2in various tissues using RT-PCR and western blotting. The results showedthat AQP2was exclusively expressed in kidney tissues. Bioinformatics analysisresults showed that3-kb pig AQP25’ fragment contains necessary componentsregulating the specific expression of AQP2. Therefore, we amplified3,090bp AQP25’-flanking sequence and cloned it into the Cre expression vector. The new Creexpression plasmid was named AQP2-Cre. The total size of the vector was11,045bp.To assess the specificity of the AQP2-Cre construct, the plasmid was transientlytransfected into PK15, Hela, A549, HepG-2and LLC-PK1cells. Initial in vitroanalysis of the AQP2-Cre plasmid was done by RT-PCR and western blotting. As aresult, the expression of the AQP2-Cre was observed specifically in the transfectedLLC-PK1cells, but not in others. After being assessed by in vitro analysis, AQP2-Cre vector was linearized usingNheI restricted enzyme and transfected into Chinese mini-pig derived fetal fibroblasts.After being cultured in selection medium containing450μg/ml G418antibiotic forapproximately14days, surviving colonies were isolated and identified by PCR. Wechose three positive cell clones in good condition as nuclear donors. A total of1427reconstructed embryos were transferred to five recipient female pigs. Two recipientsmiscarried all embryos during pregnancy and the other three produced twelve malemini-pigs. Of these mini-pigs, two died during delivery and four died soon after birth,leaving six founder piglets. Tail tissues from all12newborn cloned mini-pigs werecollected for transgene identification and fibroblast isolation. The PCR resultsrevealed that all of the mini-pigs were positive for the transgene. Sequencing resultsshowed that they all had integrated the complete Cre CDS. In addition, tail fibroblastswere isolated and frozen for future re-cloning.The Cre expression pattern in Tg mini-pigs was analyzed by RT-PCR and westernblotting. To our surprise, the Cre recombinase was uniquely expressed in all fourmini-pigs’ lung tissues instead of kidney tissues. To determine which cells specificallyexpress Cre in the lung, we performed IHC analysis on various tissues of mini-pigs.Cre staining was observed in alveolar epithelial cells. Of note, Cre was located in thenucleus and cytoplasm of epithelial cells.Subsequently, we analyzed the AQP2-Cre integration sites in Tg mini-piggenomic using inverse PCR. The results indicated that four integration sitesdistributed within four chromosomes randomly (Chr.4,12,15,18). AbsoluteqRT-PCR was used to analyze the Cre copy numbers in tail genome samples of12pigs and genome samples of various tissues. We found that the copy numbers offounders ranged from8to12. These data revealed that the copy numbers varied in thedifferent founder Tg mini-pigs, but that these differences were not significant. Inaddition, we analyzed the methylation sites in8-kb sequence of AQP2transcriptionstart sites upstream region, finding no methylation sites in test sequence.To confirm whether this Tg mini-pig line will stably express Cre recombinasewith aging, the founder Tg mini-pigs in different ages, were used to obtain lungtissues. We analyzed the Cre copies of different mini-pigs by absolute qRT-PCR. The results indicated that the copy numbers of Cre gene in different mini-pigs varied, butthe difference was not significant. Based on the results of Cre copy number analysis,we analyzed relative Cre expression levels in these founder Tg mini-pigs by relativeqRT-PCR. The data revealed that the Cre expression levels in the founder Tgmini-pigs varied over time, but not significantly.To generate F1transgenic mini-pigs, five month old Tg mini-pig was mated witha female wild-type mini-pig. Five F1mini-pigs were born in one litter. One femaleand two male mini-pigs were confirmed to be transgenic by PCR. RT-PCR andwestern blotting analysis showed that F1miniature pigs were also characterized bythe lung-specific expression. In IHC analysis, Cre staining was observed in alveolarepithelial cells. The Cre copies analysis of the F1Tg mini-pig showed only5to6Crecopies present in the different Tg mini-pigs and various tissues. The observed numberof Cre Tg copies varied, but the difference between the lowest and highest values wasnot significant. The analysis of AQP2-Cre integration sites in F1miniature pigsshowed that two contain all four sites, while the other one contains only threeintegration sites. We also analyzed the relative Cre expression changes between afounder mini-pig and its offspring. Although the genomic Cre copies between tailsfrom mini-pig No.0677and his offspring showed a significant decrease after passage,the Cre expression levels didn’t significantly change after passage. These findingssuggest that this mini-pig line can stably express Cre recombinase with aging and forgenerations.Here, the mechanisms causing unexpected expression of transgene are poorlyunderstood. Indeed, we successfully generated a lung-specific Cre expressionmini-pig tool. Importantly, the coding sequence of Cre used in this study has beenshown to be effective in previous studies and, thus, we predict that this Cre mini-pigline will be a valuable tool for investigating gene functions in alveolar epithelial cells. |
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