Dataset Information

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.

SUBMITTER: Richter A

PROVIDER: S-EPMC3537537 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Publications

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

Richter Anne A Kurome Mayuko M Kessler Barbara B Zakhartchenko Valeri V Klymiuk Nikolai N Nagashima Hiroshi H Wolf Eckhard E Wuensch Annegret A

BMC biotechnology 20121109

<h4>Background</h4>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 SCN ...[more]

PMID: 23140586

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Project description:BackgroundMelanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology.MethodFour CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis.ResultsFour vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R-/- and 10 MC3R+/- clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings.ConclusionA MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.

Dataset Information

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

Background

Results

Conclusion

Publications

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

Similar Datasets

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