Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT).

Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer's disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.

Expression pattern of a single transgene cassette located in endogenous GLIS3 of cloned pigs; a nested situation.

One of the main focus areas in transgenesis is the choice of a promoter driving stable expression over time of the gene of interest. Besides promoter identity, the genomic environment of the transgene plays a pivotal role in transcription regulation. Studies in higher mammals describing transgene expression from a defined locus are very limited. We set out to determine the expression pattern of two transgene promoters, the human PDGFß and the viral SV40, in a single cassette positioned in the largest intron of the porcine GLIS3 locus. The PDGFß promoter drives a variant of the amyloid precursor protein gene named APP695sw and the SV40 promoter drives the neomycin resistant gene, Neo. The nested gene scenario was investigated in three transgenic cloned pigs sacrificed at 3 months, 2 years and 3 years of age. With identical genetic make-up and same environment, the three individual pigs are considered representative of 3 year lifespan of a single pig. Selected organs from the pigs were analyzed by quantitative RT-PCR for transgene promoter activity as well as endogenous GLIS3 promoter activity. No apparent effect of the transgene cassette was observed on endogenous GLIS3 expression. In addition, one year old homozygous pigs showed no phenotypic signs of dysfunctional GLIS3. Both transgene promoters showed and retained their tissue specificity with stable expression over time. Our study indicates that transgenes inserted in a nested situation might be applicable for faithful and long term transgene expression.