J Vet Sci.  2014 Mar;15(1):73-80. 10.4142/jvs.2014.15.1.73.

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

• Park SK
• Roh S
• Park JI

• KMID: 1737612
• DOI: http://doi.org/10.4142/jvs.2014.15.1.73

Abstract

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.

Keyword

CDX2; EOMES; HSP70; porcine; somatic cell nuclear transfer

MeSH Terms

Animals
Biological Markers/metabolism
Cloning, Organism
Embryo, Mammalian/metabolism
Female
*Gene Expression Regulation, Developmental
Nuclear Transfer Techniques/instrumentation/*veterinary
Oocytes/metabolism
Pregnancy
Real-Time Polymerase Chain Reaction
Swine/*embryology/*genetics
Biological Markers

Figure

• 

  Fig. 1 Schematic diagram illustrating the SONT (B) and CTNT (A) protocols. The black arrowheads indicate the protruding chromosomes (oval) and first polar body (round). The black arrows indicate the nuclear donor fibroblasts (filled gray circle). SCNT: somatic cell nuclear transfer.

• 

  Fig. 2 Stereomicroscopic image of the SONT procedure. The yellow circle (A~C) indicates the oocyte chromosomes and adjacent first polar body. The blue circle (A~D) indicates the nuclear donor cell. Just after aspirating (B) and releasing (C) the oocyte chromosomes and polar body using a micropipette containing the nuclear donor fibroblast, the same pipette was used to immediately introduce the donor cell into the enucleated oocyte. Scale bars = 100 µm.

• 

  Fig. 3 Comparison of total cell numbers in blastocysts produced by SONT or CTNT. (A) Mean number of total cells in SONT and CTNT blastocysts. (B) Hoechst 33342 staining of the blastocysts (left: CTNT, right: SONT). Scale bar = 100 µm.

• 

  Fig. 4 Expression of genes related to pluripotency, trophoblast formation, metabolism, resistance to heat stress, and translation measured by real-time quantitative polymerase chain reaction. The expression of CDX2 and EOMES (two genes essential for trophectoderm formation) and HSP70 (a heat stress-associated gene) in SONT blastocysts was significantly up-regulated compared to expression levels in the control group (values in CTNT = 1; p < 0.05). Data are expressed as the mean ± SD (n = 3).

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