Evaluation of the Quality of Porcine Somatic Cell Nuclear Transfer Embryo by Gene Transcription Profiles

Abstract

Abstract. This study aimed to evaluate the quality of porcine somatic cell nuclear transfer (SCNT) embryos by examining its gene transcription patterns. Embryos were produced by SCNT, intracytoplasmic sperm injection (ICSI) or under different conditions, and transcripts of genes for fibroblast growth factor receptor (FGFr) 2IIIc, FGFr72IIIb, X inactive-specific transcript (Xist), interleukin 6 (IL6), IL6 receptor (IL6r) α and c-kit ligand, were detected by real-time RT-PCR. The percentages of embryos in which these transcripts were detected were similar in SCNT and ICSI embryos. On the other hand, the transcriptional levels of the FGFr72IIIb and IL6rα genes were 0.5 times less and 2 times more, respectively, in SCNT blastocysts than those of ICSI blastocysts (p<0.05). When nuclear transfer was performed before or after activation of oocytes, embryos in the latter case showed significantly lower frequencies of having FGFr72IIIb (74% vs. 90%) and Xist (3% vs. 33%) transcripts compared to the former case embryos (p<0.05). When two lines of nuclear donor cells with different developmental potencies were used, the transcriptional profiles in the reconstructed embryos did not show any significant differences. Our study suggests that expression profiles of FGFr72IIIb, IL6rα, and Xist can be used as markers for the diagnosis of the developmental potency of porcine nuclear transfer embryos. Key words: Nuclear transfer, Gene transcription, Embryo, Pig, Real-time RT-PCR (J. Reprod. Dev. 51: [123][124][125][126][127][128][129][130][131] 2005) n recent years, the significance of transgenic pigs has been widely recognized for biomedical uses such as organ supply for xenotransplantation, production of hybrid bioartificial organs, models for human diseases and mass production of biologically active compounds. One of the effective technologies for production of transgenic pigs, somatic cell nuclear transfer (SCNT) has been the center of attention instead of the conventional pronuclear DNA injection method [1][2][3]. With current technology, however, the rate of SCNT embryos progressing to live young is hovering between 1 and 3% or less [4]. The low birth rate of cloned pigs is the result of fetal death in early development, stillbirth and neonatal death immediately after birth [5]. These abnormalities are t h o u g h t t o a r i s e f r o m f a i l u r e i n g e n e t i c r e p r o g r a m m i n g i n t h e e a r l y s t a g e s a f t e r transplantation of embryos [6]. In other words, normal embryonic development cannot be achieved until the donor DNA is successfully reprogrammed after nuclear transfer. Factors influencing reprogramming of transferred nuclei are wide-ranging from the type and cell cycle stage

Expression of X‐linked genes in deceased neonates and surviving cloned female piglets

“…Although porcine embryos from somatic cell nuclear transfer acquire a typical methylation pattern as control embryos and have normal telomere length (Jeon et al 2005;Kang et al 2001b), they display sporatic dysregulation of genes that are critical to embryo development (Lee et al 2004;Miyazaki et al 2005). This is consistent with the widely reported abnormal gene expression in mouse and bovine NT embryos (Bortvin et al 2003;Daniels et al 2000;Han et al 2003;Mann et al 2003;Wrenzycki et al 2002;Wrenzycki et al 2001).…”

Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells

“…This finding supports a relationship between incidence of cell death and developmental potential as indicated by the lower developmental rate in NT embryos derived from FF. Aberrant gene expression is frequently observed in NT embryos of mouse, bovine, and porcine, and is one of the suggested causes of the low success rates of this approach (Boiani et al, 2002;Bortvin et al, 2003;Zhu et al, 2004;Miyazaki et al, 2005;Li et al, 2005;Tong et al, 2006). Examination of the gene expression pattern in NT embryos would aid in determining the causes of such abnormal development.…”

“…Altered levels of expression have also been detected in NT embryos when compared to their in vitro and in vivo counterparts (Wrenzycki et al, 2001). However, the amount of information gathered on the profile of gene expression in porcine NT preimplantation embryos is still scarce and limited to a handful of genes (Zhu et al, 2004;Miyazaki et al, 2005).…”

Developmental and molecular correlates of bovine preimplantation embryos

“…Determination of the expression patterns of genes relevant to early embryonic development provides an opportunity to assess the quality of the embryos produced in vitro and a chance to optimize in vitro embryo production systems. Real-time PCR is one of Molecular dynamics of bovine embryonic development 901 the most reliable methods to compare some developmentally important gene transcripts , Tesfaye et al 2004, Miyazaki et al 2005. The effects of in vitro culture conditions on gene expression have been mostly studied in later developmental stages such as morulae and blastocyst stages (Wrenzycki et al 2005).…”