J Vet Sci.  2018 Jul;19(4):492-499. 10.4142/jvs.2018.19.4.492.

Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer

• Taweechaipaisankul A
• Kim GA
• Jin JX
• Yeom SC
• Lee BC

• KMID: 2417563
• DOI: http://doi.org/10.4142/jvs.2018.19.4.492

Abstract

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.

Keyword

heterograft; miniature swine; somatic cell nuclear transfer

MeSH Terms

Adult
Cell Line*
Cytidine
DNA
Ear
Embryonic Development*
Female
Fibroblasts
Flow Cytometry
Fluorescent Antibody Technique
Genetics
Heterografts
Humans
Kidney
Korea
Physiology
Pregnancy
Skin
Swine*
Swine, Miniature
Transplantation, Heterologous
Cytidine
DNA

Figure

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  Fig. 1 Polymerase chain reaction (PCR) and real-time relative quantitative PCR results for isolated cell lines from 8 Korean native pig (KNP) piglets (No. 1–8) and skin fibroblasts of one female KNP. (A) PCR amplification products of the isolated cell lines subjected to agarose gel electrophoresis. Size of specific blood type A PCR products are approximately 340 bp. Comparison of mRNA expression levels (mean ± SEM) of FUT1 (B) and FUT2 (C). Within the same mRNA transcript, bars with asterisks indicate significantly high expression (p < 0.05). The experiment was replicated 3 times. (+), positive control; (−), negative control; M, marker; S, skin fibroblasts from female KNP; 1–8, cell lines derived from piglets No. 1–8, respectively.

• 

  Fig. 2 Immunofluorescence staining of anti-blood group antigen A in isolated cell lines from 8 Korean native pig (KNP) piglets (No. 1–8) and skin fibroblasts of one female KNP. In each sample, bright field and immunofluorescence staining under fluorescence microscopy were performed. Scale bars = 100 µm.

• 

  Fig. 3 Relative glycol-protein quantification obtained by fluorescence-activated cell sorting analysis of α 1,3-galactosyltransferase (alpha-Gal; A) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH; B) in isolated cell lines including skin fibroblasts of female Korean native pig (KNP), blood type O cells from piglets No. 1 and No. 8, and blood type A cells from piglets No. 2 and No. 3. (+), positive control (wild-type cells); (−), negative control (CMAH- and GGTA-deleted cells); 1, cell lines derived from piglet No. 1; 8, cell lines derived from piglet No. 8; S, skin fibroblasts derived from the female KNP; 2, cell lines derived from piglet No. 2; 3, cell lines derived from piglet No. 3.

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