Jannik E. Jakobsen

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

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.

Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos.

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41-42 h oocytes maturation, the oocytes were further cultured with or without 0.4 microg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 +/- 2% vs 48.1 +/- 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.

Elimination of the plasmid bacterial backbone in site-directed transgenesis

For cellular and animal transgenesis, FLP- and Cre-recombinase gene capture systems are highly effective to provide stable integration of a donor plasmid carrying the transgene cassette of interest into an engineered genomic locus in a given cell line. However, in many protocols, the entire plasmid bacterial backbone is integrated along with the transgene cassette. Here, we present a very simple yet highly efficient method for excluding plasmid bacterial backbone integration. The transgene cassette, including a single FLP recognition target site, is specifically amplified by PCR, and the resulting DNA ligated into minicircles can serve as donor DNA in FLP-mediated recombination. Interestingly, the elimination of the bacterial backbone increased expression of the inserted transgene. The presented method is simple and efficient for generating transgene cassette insertions devoid of the bacterial backbone.

Expression of the Alzheimer’s Disease Mutations AβPP695sw and PSEN1M146I in Double-Transgenic Göttingen Minipigs

Mutations in the amyloid-β protein precursor gene (AβPP), the presenilin 1 gene (PSEN1) or the presenilin 2 gene (PSEN2) that increase production of the AβPP-derived peptide Aβ42 cause early-onset Alzheimers disease. Rodent models of the disease show that further increase in Aβ42 production and earlier brain pathology can be obtained by coexpressing AβPP and PSEN1 mutations. To generate such elevated Aβ42 level in a large animal model, we produced Göttingen minipigs carrying in their genome one copy of a human PSEN1 cDNA with the Met146Ile (PSEN1M146I) mutation and three copies of a human AβPP695 cDNA with the Lys670Asn/Met671Leu (AβPPsw) double-mutation. Both transgenes were expressed in fibroblasts and in the brain, and their respective proteins were processed normally. Immunohistochemical staining with Aβ42-specific antibodies detected intraneuronal accumulation of Aβ42 in brains from a 10- and an 18-month-old pig. Such accumulation may represent an early event in the pathogenesis of Alzheimers disease.

Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig.

ABSTRACT Animal models of familial juvenile onset of Alzheimers disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation. Summary: Insight into astrocyte and radial glia pathology in an in vitro culture system derived from the APPsw pig.

Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian species suitable for locus-directed transgene expression in cell cultures and, in addition, for transgene analyses in the very early embryonic stages.

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 PDGFβ and the viral SV40, in a single cassette positioned in the largest intron of the porcine GLIS3 locus. The PDGFβ 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.

Pancreas specific expression of oncogenes in a porcine model

Pancreatic cancer is the fourth leading course of cancer death and early detection of the disease is crucial for successful treatment. However, pancreatic cancer is difficult to detect in its earliest stages and once symptoms appear, the cancer has often progressed beyond possibility for curing. Research into the disease has been hampered by the lack of good models. We have generated a porcine model of pancreatic cancer with use of transgenic overexpression of an oncogene cassette containing MYC, KRASG12D and SV40 LT. The expression was initiated from a modified Pdx-1 promoter during embryogenesis in a subset of pancreatic epithelial cells. Furthermore, cells expressing the oncogenes also expressed a yellow fluorescent protein (mVenus) and an inducible negative regulator protein (rtTR-KRAB). Cells where the Pdx-1 promoter had not been activated, expressed a red fluorescent protein (Katushka). In vitro analyses of cells obtained from the transgenic pigs showed increased proliferation and expression of the transgenes when activated. Induction of the repressor protein eliminated the oncogene expression and decreased cell proliferation. In vivo analysis identified foci of pancreatic cells expressing the oncogenes at day zero post farrowing. These populations expanded and formed hyperplastic foci, with beginning abnormality at day 45. Cells in the foci expressed the oncogenic proteins and the majority of the cells were positive for the proliferation marker, Ki67. We predict that this model could be used for advanced studies in pancreatic cancer in a large animal model with focus on early detection, treatment, and identification of new biomarkers.