Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

Abstract

Background: Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs) as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination.

Results: PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs) and porcine ear fibroblasts (PEFs) could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT.

Conclusion: The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

Figure 1

Single cell clone colonies of PKCm at P3. Single cell colonies were generated and analyzed after 7 days. The cells and formed colonies differed morphologically: fibroblast-like cells [A, B, D-F, H], epithelial- and endothelial-like cells [C, G], cell size (large [B], smaller [A, C-H]), colony compactness (cells very close [A, E, G], gaps between cells [B, C, D, F, H]) and colony shape (clearly defined [A, C, E, G], frayed colonies [B, D, F, H]). Scale bar = 100 μm.

Figure 2

Morphology of different primary pig cell lines after several passages. Porcine kidney cells (PKCm and PKC2109), fetal fibroblasts (PFF26) and ear fibroblasts (PEF0110) showed all a more or less heterogeneous composition. In all primary cell lines the morphology changed from smaller and round-shaped in early passage (P2) to larger and long spindle-shaped cells in later passage (P13). Scale bar = 100 μm.

Figure 3

Growth potential of porcine kidney cells compared to fetal and ear fibroblasts. PKCm (A, left and middle panel) and PKC2109 (B, left and middle panel) grew rather in islands at P4 on non-coated plates compared to collagen-coated plates where they grew evenly spread. Scale bar = 100 μm. The proliferation assay showed best growth potential on collagen-coated plates with PKCm (A, right diagram) and PKC2109 (B, right diagram). (C) Growth curves of PKCm, PKC2109, PFF26 and PEF0110. Every 12–24 h cell numbers of various cell types were determined. The x-section (exponential growth phase) was used for calculation of population doubling time. PKC2109 and PKCm exhibited the shortest population doubling time compared to PFF26 and PEF0110. (D) Correct karyotype (2n=38) of PKC2109 at passage 71.

Figure 4

Transiently nucleofected porcine kidney cells. PKCm (A) and PKC2109 (B) 24 h after nucleofection with a GFP expressing plasmid using 0.5×10⁶ cells and 2 μg DNA at passage 4. Scale bar = 100 μm.

Figure 5

Generation of transgenic pigs by additive gene transfer and gene targeting combined with SCNT. (A) Additive gene transfer. Cells are transfected with a vector containing the gene of interest (GOI) and a resistance gene. After antibiotic selection the single cell clones are mixed and transferred to an enucleated oocyte (SCNT) followed by an embryo transfer (ET) to synchronized gilts. After genotyping and gene expressing analysis of the born pigs the best expressing animal is re-cloned or used for a second round of transfection. (B) Gene targeting. In the first targeting round the cells are transfected with a targeting vector containing beside homologous regions of the target locus, a resistance gene (RG1). After selection and characterization of the generated single cell clones, heterozygous knockout cells are used for SCNT followed by the ET. For the targeting of the second allele cells isolated of the heterozygous knockout animals are used for a second round of transfection, using a second targeting vector with another resistance gene (RG2). All following procedures resemble the first round.