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

Anne Richter,Mayuko Kurome,Barbara Kessler,Annegret Wuensch,Valeri Zakhartchenko,Nikolai Klymiuk,Hiroshi Nagashima,Eckhard Wolf

doi:10.1186/1472-6750-12-84

Anne Richter, Mayuko Kurome + Show 6 more

Open Access

https://doi.org/10.1186/1472-6750-12-84

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Abstract

BackgroundSomatic 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.ResultsPKCs 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.ConclusionThe 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.

Highlights

Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research
We investigated porcine fetal fibroblasts (PFFs) and ear fibroblasts (PEFs) which displayed morphological heterogeneity, though to a lesser extent than porcine kidney cells (PKCs)
In the primary cell lines PKCm, PKC2109, PFF26 and PEF0110, the heterogeneous appearance diminished with increasing passage numbers and the culture became dominated by cells with spindle shaped fibroblast-like morphology (Figure 2)

Summary

Introduction

Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. Over the last years the pig is getting more and more attractive as model organism for biomedical research due to similarities with humans in anatomy, size, physiology, metabolism and pathology (reviewed in [1]). Genetic modification of pigs can be achieved by a variety of approaches, including pronuclear microinjection of DNA, sperm-mediated gene transfer, retroviral transduction, and somatic cell nuclear transfer (SCNT) using transfected donor cells (reviewed in [4,5]). SCNT using pools of stable transfected cell clones was an efficient way for the production of transgenic founder pigs with appropriate expression patterns (reviewed in [1,9]) and facilitated the generation of the first pig models with inducible transgene expression [10]

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